Exendin-4 analogue pegylated with polyethylene glycol or derivative thereof, preparation method thereof, and pharmaceutical compostion for preventing or treating diabetes, containing same as active ingredient

ABSTRACT

The present disclosure relates to an exendin-4 analogue PEGylated with polyethylene glycol or a derivative thereof, a preparation method, and a pharmaceutical composition for prevention or treatment of diabetes containing the same as an active ingredient. According to the present invention, the yield of an exendin-4 analogue can be increased via the selective PEGylation by using exendin-4 in which a cysteine is introduced into #40 site of the C-terminal, and treatment effect of medications can be increased, so that the exendin-4 analogue can be usefully applied as a composition for prevention or treatment of diseases caused by insulin hypersecretion.

TECHNICAL FIELD

The present disclosure relates to an exendin-4 analogue PEGylated withpolyethylene glycol or a derivative thereof, a preparation methodthereof, and a pharmaceutical composition for prevention or treatment ofdiabetes containing the same as an active ingredient.

BACKGROUND ART

Among pharmaceutical technologies, PEGylation of peptides and proteinsfor the purpose of treatment is the most effective technology.PEGylation of peptides and proteins increases molecular weight thereof,protein degradation site defense and immunogenicity site defense, whichconsequently increases half-life of in vivo medications and reduceimmunogenicity of peptides and proteins. Therefore, PEGylationtechnology has an effect of increasing treatment effect by solvingproblems of original medications, and due to such strength, serves animportant role in increasing effects of PEGylated peptide and proteinmedication delivery system.

Also, peptides and proteins increase treatment effect by covalentlybonding with polyethylene glycol (PEG). Such technology increasesmolecular weight, defense of a metabolism site and inhibition of animmunogenicity site, increasing in vivo half-life and stability andreducing immunogenicity. Furthermore, kidney excretion of peptides andproteins bound with PEG is reduced due to the increase of molecularweights of peptides and proteins by PEG, so that PEGylation hasadvantages of increasing effects in both pharmacokinetically andpharmacodynamically.

PEGylation reacting sites of peptides and proteins are randomlydispersed and are occasionally close to bioactive sites. However,traditional PEGylation employs nonspecific PEGylation methods that donot consider PEG reacting site, number of PEG bonds and biologicalactivity. However, such a nonspecific PEGylation method reducestreatment effects by bringing insufficient conformation by producingvarious branched type PEG-bonded isomers that have differentphysiochemical, biological and pharmacokinetic characteristics. SpecificPEGylation methods have been studied to solve such problems, andrecently the specific PEGylation methods are rapidly developing tobecome a method of maximizing medications' treatment effects as geneticengineering technology and selective functional group introducingtechnology are quickly developing. In a related art, a study ofselectively binding PEG into N-terminal site after removing a reactionsite by substituting primary amine site with different amino acid usinggenetic engineering method for granulocyte stimulating factor (G-CSF)and tumor necrosis factor receptor has been conducted previously.

Also, studies using a technology that selectively PEGylates substituentafter having introduced a specific substituent using genetic engineeringmethods and substitution technology for medications such asstaphylokinase, interferon a-2, antibody single chain fragment variable(ScFv), have been conducted.

Exendin-4 is a polypeptide substance and is the first incretin analogue,a diabetes medication prepared by synthesizing exendin-4, a salivarysubstance of Gila monster. Exendin-4 is different from exendin-3 foronly #2 and #3 sites, is known to have a longer half-life than glucagonlike peptide-1 (GLP-1) which is a diabetes medication having a half-lifeshorter than two minutes for DPP-IV, an enzyme that is resistant fordirectly degrading incretin enzyme that is produced in mammals' stomachsafter ingestion by DPP-IV (dipeptidyl peptidase-4) to serve beneficialroles of promoting insulin secretion and lowering blood sugar level, andalso, it shows 2-4 hours of half-life in vivo experiment, and it hasbeen confirmed that it can reach enough blood concentration with 2-3times of intraperitoneal injection per day.

Also, exendin-4 is known to control gastrointestinal tracts' motility,reduces food intake and suppresses blood plasma glucagon, and recentlyPLGA microsphere type synthetic exendin-4 (product name: Byetta) hasbeen authorized by US FDA and is about to be released. However, sincethis Byetta LAR product has complicated preparation process and is shortin vivo half-life for exendin-4, which is about 4-6 hours, frequentadministration of high dose exendin-4 is required, and the problem ofmedication release control based on quick excretion due to the lowmolecular weight of lower than 4200, and problems such as immunogenicitystill exist.

Therefore, while studying a method to reduce administration frequency ofexendin-4 and solve the low molecular weight problem of exendin-4, theinventors have completed the present invention after having confirmedthe fact that it is possible to increase the production yield ofPEGylated exendin-4 and treatment effect of medications by performingselective PEGylation via insertion of cysteine (Cys) amino acid into thesite (#40 site) next to #39 site of C-terminal of exendin-4.

DISCLOSURE OF THE INVENTION Technical Problem

One object of the present invention is to provide an exendin-4 analoguein which a cysteine (Cys) is introduced into #40 site of C-terminal andis PEGylated with polyethylene glycol (PEG) or derivatives thereof.

Another object of the present invention is to provide a method ofpreparing the exendin-4 analogue.

Still another object of the present invention is to provide apharmaceutical composition for prevention or treatment of diseasescaused by insulin hypersecretion, containing the exendin-4 analogue asan active ingredient.

Technical Solution

In order to achieve the objects, the present invention provides anexendin-4 analogue that has a cysteine (Cys) introduced into #40 site ofC-terminal, which is PEGylated with polyethylene glycol (PEG) orderivatives thereof.

The present invention also provides a method of preparing the exendin-4analogue.

Furthermore, the present invention provides a pharmaceutical compositionfor prevention or treatment of diseases caused by insulin hypersecretioncontaining the exendin-4 analogue as an active ingredient.

Advantageous Effects

According to the present invention, by performing selective PEGylation,the yield of an exendin-4 analogue in which a cysteine (Cys) isintroduced into #40 site of the C-terminal and is PEGylated withpolyethylene glycol (PEG) or derivatives thereof, can be increased, andtreatment effect of medications can be increased, and thus the exendin-4analogue can be beneficially used as a composition for prevention ortreatment of diseases caused by insulin hypersecretion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view illustrating PEGylation of exendin-4 in whichcysteine (Cys 40) is introduced into the C-terminal of example 1 of thepresent invention.

FIG. 2 is a schematic view illustrating PEGylation for lycine amine ofexendin-4 of comparative example 1 of the present invention.

FIG. 3 is a schematic view illustrating PEGylation for N-terminal ofexendin-4 of comparative example 2 of the present invention.

FIG. 4 is a view illustrating light absorbance of example 1 of thepresent invention.

FIG. 5 is a view illustrating light absorbance of comparative examples1a to 1c of the present invention.

FIG. 6 is a view illustrating light absorbance of comparative example 2of the present invention.

FIG. 7 is a view illustrating the production yield of example 1 of thepresent invention.

FIG. 8 is a drawing illustrating product yield of comparative examples1a to 1c of the present invention.

FIG. 9 is a view illustrating the production yield of comparativeexample 2 of the present invention.

FIG. 10 is a view illustrating the affinity of a PEG bound exendin-4analogue to a GLP-1 receptor according to an example of the presentinvention.

FIG. 11 is a schematic view illustrating PEG bound exendin-4 analoguesof examples 4 and 5 of the present invention.

FIG. 12 is a view illustrating blood glucose level for diabetic miceadministrated with a PEG bound exendin-4 analogue according to anexample of the present invention.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention provides an exendin-4 analogue in which a cysteine(Cys) is introduced into #40 site of the C-terminal and is PEGylatedwith polyethylene glycol (PEG) or a derivative thereof.

The molecular weight of polyethylene glycol or a derivative thereofaccording to the present invention is 5-60 kDa, and preferably 20-50kDa, but is not limited thereto.

Also, the polyethylene glycol or a derivative thereof according to thepresent invention is a linear type or a branched type, and for thebranched type, preferably a dimeric type or a trimeric type may be used,and more preferably a trimeric type may be used.

Specifically, the polyethylene glycol derivative is, for example,methoxypolyethylene glycol succinimidylpropionate, methoxypolyethyleneglycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde,methoxypolyethylene glycol maleimide, or multiple branched types ofthese derivatives. Preferably, the polyethylene glycol derivative islinear methoxypolyethylene glycol maleimide, branch typemethoxypolyethylene glycol maleimide or trimeric methoxypolyethyleneglycol maleimide, and more preferably is trimeric methoxypolyethyleneglycol maleimide.

Also, the present invention provides a method of preparing an exendin-4analogue PEGylated with the polyethylene glycol or a derivative thereof,which includes a process of dissolving exendin-4 in which cysteine isintroduced into #40 site of the C-terminal, and polyethylene glycol or aderivative thereof in phosphate buffer saline solution and reacting themat room temperature.

Specifically, an exendin-4 analogue PEGylated with polyethylene glycolor a derivative thereof may be prepared by adding exendin-4 in whichcysteine is introduced into #40 site of the C-terminal, and polyethyleneglycol or a derivative thereof in a phosphate buffer saline solution ina mole ratio of 1:1-3 in a phosphate buffer saline having a pH range of7.2-7.8, preferably pH 7.5, dissolving these ingredients, and perform areaction for 1-3 hours at room temperature although the reactiontemperature is not particularly limited, and performing a columnchromatography after the reaction is completed.

When the phosphate buffer saline is not within the pH range, the yieldmay decrease.

In the present invention, after the exendin-4 analogue PEGylated withpolyethylene glycol or the derivative thereof is prepared, the molecularstructure of the exendin-4 analogue may be confirmed by a massspectroscope, a liquid chromatography, an X-ray diffraction analysis, apolarimetry, and comparison between calculated values and measuredvalues of representative elements constituting the exendin-4 analogue.

Also, the present invention provides a pharmaceutical composition forprevention or treatment of diseases caused by insulin hypersecretion,containing the exendin-4 analogue as an active ingredient.

Furthermore, the present invention provides a treatment methodcharacterized with administration of the exendin-4 analogue PEGylatedwith polyethylene glycol or a derivative thereof to patients in need oftreating the diseases caused by insulin hypersecretion.

The diseases caused by insulin hypersecretion may include Type 1diabetes, Type 2 diabetes and diabetes complications.

As a result of having measured affinity to a GLP-1 receptor of exendin-4analogue PEGylated with polyethylene glycol or a derivative thereofaccording to the present invention, IC50 value was 1.04 nM, and this wasconfirmed to show 120 times more activity than compound of example 1(Nter-PEG-Ex4) (IC50=121. 78 nM) (refer to experimental example 1, Table3 and FIG. 10).

Also, for better understanding, a schematic diagram of the presentinvention's exendin-4 bound with a trimeric PEG at C40 site is shown inFIG. 11.

When the molecular weight of the bound PEG is 23K, PEG of 3KD is used asa PEG spacer, and PEG having 10KD molecular weight are bound to terminalof the 3KD (example 4). Also, similar to this, when the molecular weightof the bound PEG is 50, PEG of 10KD is used as a PEG spacer, and PEGhaving the molecular weight of 20KD are bound to terminal of the 10KD(example 5). At this time, as a result of having measured the requiredtime of blood glucose level raising back to 8.35 mmol/L after havinginjected the exendin-4 of example 4 (C40-PEG23K-Ex4) and example 5(C40-PEG50K-Ex4), low blood glucose level maintained from 45.5-56.1hours after the administration of the medication (refer to experimentalexample 2, Table 4 and FIG. 12), which was confirmed to be more thantwice of C40-PEG20K-Ex4 (23.2 hours) and control group (7.3 hours),enabling 7-8 times more stable maintenance of blood glucose level.

Therefore, the C40 site specific PEG bound exendin-4 compound accordingto the present invention can solve the drawback of quick excretion ofmedications due to the low molecular weight of existing exendin-4, hasexcellent affinity to the GLP-1 receptor, and has strong low bloodglucose maintaining ability capable of maintaining blood glucose levelup to 3-4 days after having administrated the medications, so it can beused beneficially for preventing or treating insulin hypersecretionrelated Type 1 diabetes, Type 2 diabetes and diseases related withdiabetes complications.

When the composition of the present invention is used as medications,the pharmaceutical composition containing the exendin-4 analoguePEGylated with polyethylene glycol or a derivative thereof may beadministrated after having formulated into various oral or non-oraladministration forms as the following in case of clinicaladministration, but is not limited thereof.

For oral administration purposed formulation, for example, there aretablets, pellets, hard/soft capsules, liquids, suspensions, emulsifiers,syrups, granules, elixirs, troches, etc., and these formulations includediluents (example: lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine), slip modifiers (example: silica, talc,stearate and its magnesium or calcium salt and/or polyethylene glycol)in addition to the active ingredient. Tablets may also include binderssuch as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethyl cellulose and/or polyvinyl pyrrolidine,and may include disintegrating agents such as starch, agar, alginic acidor sodium salt thereof or boiling mixture and/or absorbents, coloringagents, flavoring agents and sweetening agents if needed.

The pharmaceutical composition containing the exendin-4 analoguePEGylated with polyethylene glycol or a derivative thereof may benon-orally administrated, and the administration is done by subcutaneousinjection, intravenous injection, intramuscular injection orintrathoracic injection.

At this time, the exendin-4 analogue PEGylated with polyethylene glycolor a derivative thereof may be may be prepared into liquid or suspensionby having mixed it with stabilizer or buffer in water to formulize itinto non-orally administration purposed formulation, and this may beprepared into ampoule or vial unit administration form. The compositionis sterilized and/or may include adjuvants such as antiseptics,stabilizers, hydrators or emulsify stimulators, osmotic pressurecontrolling purposed salts and/or buffers, and other substancesbeneficial for treatments, and may be formulated according totraditional methods of mixture, granulation or coating.

The human body dose of the pharmaceutical composition containing theexendin-4 analogue PEGylated with polyethylene glycol or a derivativethereof according to the present invention may vary depending on theage, body weight, gender, administration form, health status and levelof disease of patients, and may be administrated via oral or non-oralroute following decisions of doctors or pharmacists with preferably doseof 0.01 to 200 mg/kg/day.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail by examples andexperimental examples hereafter.

The examples and experimental examples are only demonstrating thepresent invention, and the contents of the present invention are notlimited thereof.

Examples 1-5 C40 Site Specific PEG Bound Exendin-4 Production

To prepare C40 site specific PEG bound exendin-4, exendin-4-Cys in whichcysteine is introduced into the C-terminal site (#40 site) was used(exendin-Cys, molecular weight: 4290.7, sequence:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC), and maleimide activatedmonomethoxy PEG (mPEG-MAL, MW: 5, 20 kDa(Linear type), 20 kDa (Branchtype), 23, 50 kDa (Trimer type)) was purchased from Nippon Oil and Fats,NOF, Tokyo, and used.

To prepare C-terminal #40 site specific PEG bound exendin-4,exendin-4-Cys and mPEG-MAL (MW: 5, 20 (linear type), 20 (branch type),23, 50 kDa) were completely dissolved in a mole ratio of 1:2 in a 20 mMphosphate buffer saline (pH 7.5) and were reacted for two hours at roomtemperature (refer to FIG. 1). After the reaction, the reacted solutionwas separated by a reversed phase chromatography with Capcell-pak RP-18column (250×10 mm, 5 μm, Shiseido, Japan) at a flow speed of 5.0 ml/min.The separation was monitored at 215 nm wavelength ultraviolet ray. Themobile phase was separated using a linear concentration gradient method(36-42% B over 30 min) for 0.1% TFA distilled water (mobile phase A) and0.1% TFA acetonitrile (mobile phase B) (refer to FIG. 4).

The peaks separated by the method were collected separately,acetonitrile was removed using nitrogen gas, and the removed solutionwas concentrated using Centricon-10 (Mw cut off 3000, Millipore Corp.,Billerica, Mass.). The prepared substance was stored at 4° C. andprepared by mixing 1 μl sample-matrix sample solution and 2 μl matrixsolution, and the matrix solution was prepared by dissolvingα-cyanohydroxycinnamic acid (α-CHCA) with water/CAN (50:50) solutioncontaining 0.1% (v/v) TFA. The prepared 1 μl sample-matrix solution wasput on a sample plate, dried at vacuum status and analyzed with sizeexclusion chromatography (SEC) and MALDI-TOF mass spectrometer, and C40site specific PEG bonding reaction (C40-PEG-Ex4) was analyzed at 0, 20,40, 60 and 80 minutes and was shown with chromatogram area ratio incomparison with the initial status of exendin-4 and C40-PEG-Ex4. Theresult is illustrated in Table 1 and FIG. 7.

TABLE 1 Reaction Time Yield (%) Example 1 80 min. 93% C40-PEG_(5K)-Ex4(linear) Example 2 80 min. 89% C40-PEG_(20K)-Ex4 (linear) Example 3 80min. 91% C40-PEG_(20K)-Ex4 (branch) Example 4 80 min. 90%C40-PEG_(23K)-Ex4 (trimer) Example 5 80 min. 85% C40-PEG_(50K)-Ex4(trimer)

As shown in Table 1, the reaction time was 80 minutes in average,production being done with yield of over 90% average (refer to FIG. 7).

Comparative Example 1 Production of Non-Specific PEG Bound Exendin-4

A method equivalent to the example 1 except for using exendin-4(molecular weight: 4186.6, sequence:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS) and succinimidyl activatedmonomethoxy PEG (mPEG-SPA, MW: 5, 20 kDa (Linear type)) instead of usingcysteine introduced exendin-4-Cys and maleimide activated monomethoxyPEG, was performed to prepare non-specific PEG bound exendin-4 (refer toFIG. 2 and FIG. 5).

The succinimidyl activated monomethoxy PEG (mPEG-SPA) was purchased fromNippon Oil and Fats, NOF, Tokyo, and used.

TABLE 2 Reaction Comparative example 1 Time Yield (%) Comparativeexample 1a 80 min. 20% Lys¹²-PEG_(20K)-Ex4 Comparative example 1b 80min. 31% Lys²⁷-PEG_(20K)-Ex4 Comparative example 1c 80 min. 25%Lys^(12,27)-PEG_(20K)-Ex4

As shown in Table 2, the reaction time of non-specific primary amine PEGbinding reaction was 80 minutes in average, average yield being 20% forComparative example 1a(Lys¹²-PEG_(20K)-Ex4) and 31% for Comparativeexample 1b(Lys²⁷-PEG_(20K)-Ex4) (refer to FIG. 8).

Comparative Example 2 N-Terminal Specific PEG Bound Exendin-4 Production

A method equivalent to the example 1 except for using exendin-4(molecular weight: 4186.6, sequence:HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS) and monomethoxy PEG-aldehyde(mPEG-ALD, MW: 5 kDa (linear)) instead of using cysteine introducedexendin-4-Cys and maleimide activated monomethoxy PEG, was performed toprepare non-specific PEG bound exendin-4 (refer to FIG. 3 and FIG. 6).

The monomethoxy PEG-aldehyde was purchased from Nippon Oil and Fats,NOF, Tokyo and used.

As a result, the reaction time of N-terminal specific PEG bindingreaction (N_(ter)-PEG_(5K)-Ex4) was 720 minutes, with average yield of72% (refer to FIG. 9).

Experimental Example 1 Analysis of RIN-m5F Cell Receptor BindingAffinity of PEG Bound Exendin-4 Analogue

The following experiment was performed to perform GLP-1 receptor(GLP-1R) affinity of PEG bound exendin-4 analogues of Example 1(C40-PEG_(5K)-Ex4), Comparative example 1a (Lys¹²-PEG_(5K)-Ex4),Comparative example 1b (Lys²⁷-PEG_(5K)-Ex4) and Comparative example 2(N_(ter)-PEG_(5K)-Ex4) prepared in Example 1, Comparative example 1 and2.

Islet cells (RIN-m5F, ATCC, Manassas, Va.) expressing vast quantity ofGLP-1 receptor (GLP-1R) were inoculated in 12-wells plates. It waswashed twice with binding buffer (120 mM NaCl, 1.2 mM MgSO₄, 13 mMsodium acetate, 5 mM KCl, 1.2 g/l Tris, 2 g/l bovine serum albumin, 1.8g/l glucose, pH 7.6) after 48 hours and unmarked PEG bound exendin-4analogue (final concentration range: 0.001-1000 nM) and exendin-4 markedwith 30 pM concentration I-125 (9-39, PerkinElmer, Boston, Mass.) weretreated simultaneously. Thorough washing was done with PBS including 1mg/l bovine serum albumin after two hours. Finally the cells werethoroughly degraded for 15 minutes using cell lysis buffer (0.5 N NaOHwith 1% SDS), and the radiation level of I-125 was measured using agamma counter (GMI, Inc., Ramsey, Minn.). The result is illustrated inTable 3 and FIG. 10.

TABLE 3 IC₅₀ (nM) Example 1 1.04 nM (C40-PEG_(5K)-Ex4) Comparativeexample 1a 6.45 nM (Lys¹²-PEG_(5K)-Ex4) Comparative example 1b 2.42 nM(Lys²⁷-PEG_(5K)-Ex4) Comparative example 2 121.78 nM (N_(ter)-PEG_(5K)-Ex4) Control group 0.23 nM (exendin-4)

As shown in Table 3, IC₅₀ of Example 1 (C40-PEG_(5K)-Ex4) according tothe present invention was confirmed to be 1. 04 nM after the affinityfor GLP-1 receptor was measured. It was confirmed that it shows activitytwice better than Comparative example 1b (Lys²⁷-PEG_(5K)-Ex4)(IC₅₀value=2.42 nM), and 6 times better than Comparative example 1a(Lys¹²-PEG_(5K)-Ex4) (IC₅₀ value=6.45 nM). Also, it was confirmed thatExample 1 (C40-PEG_(5K)-Ex4) according to the present invention showsactivity 120 times better than Comparative example 2(N_(ter)-PEG_(5K)-Ex4) (IC₅₀value=121.78 nM).

Therefore, C40 site specific PEG bound exendin-4 composition accordingto the present invention not only can solve the weakness of quickexcretion of medications due to low molecular weight of exendin-4, butalso can be used beneficially as a diabetes medication since GLP-1receptor affinity shows similar biological activity as exendin-4 (referto FIG. 10).

Experimental Example 2 Evaluation of Low Blood Glucose Sustainability inNon-Fasting Type 2 Diabetic Mice

The following experiment was performed to evaluate low blood glucosesustainability of C40 site specific PEG bound exendin-4 compositionaccording to the present invention in Type 2 diabetic mice.

Type 2 diabetic C57BL/6 db/db mice (male, 4-5 weeks old, Central Lab.Animal Inc.) were used, and animals were exposed to light at 12 hourscycle and were grown after having stabilized two week by allowing freeintake of foods and water. The experimental animals were managedfollowing the guideline of National Institute of Health (NIH) andauthorized by Institutional Animal Care and Use Committee ofSungkyunkwan University, and the experiment was performed humanely.

C40-PEG_(5K)-Ex4 (linear), C40-PEG-Ex4 (linear), C40-PEG_(20K)-Ex4(branch), C40-PEG_(23K)-Ex4 (trimer) and C40-PEG_(50K)-Ex4 (trimer)prepared from the Example 1 to 5 and Lys²⁷-PEG_(20K)-Ex4 prepared inComparative example 1 b were intraperitoneally injected with 25 nmol/kgdose to male db/db mice (6-7 weeks old), blood was collected from tailvein of mice following the float time:0, 0.5, 1, 2, 3, 4, 6, 8, 12, 24,36, 48, 60, 72, 96 hours and blood glucose concentration was measuredwith ACCU-CHEK Sensor (Roche Diagnostics Corp., USA). Afterwards, thelow blood glucose sustaining time (blood glucose level <8.35 mmol/l (150mg/dL)) was additionally measured and shown in Table 4 and FIG. 12. Inthe present experiment, exendin-4 was used as the control group.

TABLE 4 Blood glucose level (mmol/l) (average) Comparative C40-PEG-Ex4Example 1b Control Time Example 1 Example 2 Example 3 Example 4 Example5 (Lys²⁷- group Untreated (h) (PEG_(5K)) (PEG_(20K)) (PEG_(20K))(PEG_(23K)) (PEG_(50K)) PEG_(20K)-Ex4) (Ex-4) group 0 23.38 24.28 24.4424.22 24.56 24.13 22.61 24.23 0.5 7.62 7.96 7.86 7.97 7.63 7.97 6.9524.21 1 7.36 6.13 6.89 6.99 6.25 6.56 6.41 23.44 2 5.09 5.29 5.04 4.965.45 5.24 5.80 24.58 3 4.46 4.18 4.15 4.11 4.64 4.22 5.85 22.96 4 4.934.34 4.66 4.54 4.23 4.29 8.02 24.54 6 5.73 4.9 4.67 4.87 4.26 4.85 10.6923.43 8 9.04 4.57 5.11 4.66 4.69 5.13 16.01 24.94 12 16.2 5.86 7.89 4.94.87 5.40 23.89 22.47 24 21.1 8.54 15.09 5.52 5.11 12.98 — 24.42 36 —11.47 20.14 8.08 6.31 17.34 — 23.92 48 — 15.34 24.21 8.66 7.26 20.45 —22.66 60 — 20.45 23.76 11.34 8.87 23.02 — 23.41 72 — 23.02 — 14.12 13.49— — 22.26 96 — — — 18.79 17.07 — — 24.51 120 — — — 24.53 23.02 — — 23.75

As shown in Table 4, the time required for blood glucose level of C40site specific PEG bound exendin-4 of Examples 1 to 5 according to thepresent invention increasing back to 8.35 mmol/l was confirmed to belonger than exendin-4 (7.3 hours), and especially for Example4(C40-PEG_(23K)(-Ex4) and Example 5 (C40-PEG_(50K)-Ex4) which wereintroduced with trimer PEG, the low blood glucose level sustained for45.5 hours and 56.1 hours, respectively (refer to FIG. 12).

Therefore, C40 site specific PEG bound exendin-4 composition accordingto the present invention can be used beneficially as a diabetesmedication by having solved the weakness of quick excretion ofmedications due to low molecular weight of exendin-4 and consequentlysustaining blood glucose level 7-8 times more stable than theComparative examples.

Meanwhile, C40 site specific PEG bound exendin-4 analogue according tothe present invention can be formulated into various forms followingpurposes. The following is an illustration of few formulation methodsthat include C40 site specific PEG bound exendin-4 analogue according tothe present invention as an active ingredient, and the present inventionis not limited thereof.

Formulation Example 1 Production of Powders

C40 site specific PEG bound exendin-4 analogue 2 g Lactose 1 g

The ingredients were mixed and stuffed in sealed packages to preparepowders.

Formulation Example 2 Production of Tablets

C40 site specific PEG bound exendin-4 analogue 100 mg Corn starch 100 mgLactose 100 mg Magnesium stearate  2 mg

The ingredients were mixed and compressed according to generalpreparation methods for tablets to prepare tablets.

Formulation Example 3 Production of Capsule

C40 site specific PEG bound exendin-4 analogue 100 mg Corn starch 100 mgLactose 100 mg Magnesium stearate  2 mg

The ingredients were mixed and stuffed in gelatin capsules according togeneral preparation methods for capsules to prepare capsules.

Formulation Example 4 Production of Injections

C40 site specific PEG bound exendin-4 analogue 100 mg Mannitol 180 mgNa₂HPO₄•2H₂O  26 mg Distilled water 2974 mg 

The ingredients were included with the given quantity according togeneral preparation methods for injections to prepare injections.

INDUSTRIAL APPLICABILITY

According to the present invention, by performing selective PEGylation,the yield of an exendin-4 analogue in which a cysteine (Cys) isintroduced into #40 site of the C-terminal and is PEGylated withpolyethylene glycol (PEG) or a derivative thereof, can be increased andthe treatment effect of medications can be increased, and thus theexendin-4 analogue can be beneficially used as a composition forprevention or treatment of diseases caused by insulin hypersecretion.

We claim:
 1. An exendin-4 analogue wherein a cysteine (Cys) isintroduced into #40 site of the C-terminal and is PEGylated withpolyethylene glycol (PEG) or a derivative thereof.
 2. The exendin-4analogue as set forth in claim 1, wherein, the polyethylene glycol orthe derivative thereof is a linear type or a branched type.
 3. Theexendin-4 analogue as set forth in claim 1, wherein, the polyethyleneglycol or the derivative thereof is a dimeric type or a trimeric type.4. The exendin-4 analogue as set forth in claim 3, wherein, thepolyethylene glycol or the derivative thereof is the trimeric type. 5.The exendin-4 analogue as set forth in claim 1, wherein, thepolyethylene glycol or the derivative thereof has a molecular weight of5-60 kDa.
 6. The exendin-4 analogue as set forth in claim 5, wherein,the polyethylene glycol or the derivative thereof has a molecular weightof 20-50 kDa.
 7. The exendin-4 analogue as set forth in claim 1,wherein, the polyethylene glycol derivative is methoxypolyethyleneglycol succinimidylpropionate, methoxypolyethylene glycolN-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde,methoxypolyethylene glycol maleimide, or multiple branched types of thederivatives.
 8. The exendin-4 analogue as set forth in claim 7, wherein,the polyethylene glycol derivative is selected from the group consistingof linear methoxypolyethylene glycol maleimide, dimericmethoxypolyethylene glycol maleimide or trimeric methoxypolyethyleneglycol maleimide.
 9. The exendin-4 analogue as set forth in claim 8,wherein, the polyethylene glycol derivative is trimericmethoxypolyethylene glycol maleimide.
 10. A method of preparing theexendin-4 analogue as set forth in claim 1, the method comprising:dissolving exendin-4 in which a cysteine is introduced into #40 site ofthe C-terminal, and polyethylene glycol or a derivative thereof in aphosphate buffer saline solution; and reacting the dissolved ingredientsat room temperature.
 11. The method as set forth in claim 10, wherein,the phosphate buffer saline has a pH range of 7.2-7.8.
 12. The method asset forth in claim 10, wherein, the reaction mole ratio of the exendin-4having the introduced cysteine and the polyethylene glycol or thederivative thereof is 1:1-3.
 13. A pharmaceutical composition forprevention or treatment of diseases caused by insulin hypersecretion,containing an exendin-4 analogue as set forth in claim 1 as an activeingredient.
 14. The pharmaceutical composition as set forth in claim 13,wherein, the diseases caused by insulin hypersecretion are Type 1diabetes, Type 2 diabetes or diabetes complications.